Tyrosine Hydroxylase Overexpression Lysate

Western Blot: Tyrosine Hydroxylase Overexpression Lysate [NBP2-11149] - (Adult Normal) [NBP2-11149] Left lane: Empty vector transfected control cell lysate (HEK293 cell lysate); Right lane: Over-expression Lysate for Tyrosine Hydroxylase.

• Reactivity: Hu
• Applications: WB

Tyrosine Hydroxylase Overexpression Lysate Summary

• Description:

  Tyrosine Hydroxylase Transient Overexpression Lysate

  
  Expression Host: HEK293T
  
  Plasmid: RC211218
  
  Accession#: NM_000360
  
  Protein Tag: C-MYC/DDK
  
  You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul). Each vial of cell lysate contains 100ug of total protein (at 1 mg/ml). The 2xSDS Sample Buffer consists of 4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT.
• Gene: TH

Applications/Dilutions

• Dilutions:
  • Western Blot
• Application Notes: This product is intended for use as a positive control in Western Blot. Overexpression of the target protein was confirmed using an antibody to DDK (FLAG) epitope tag (NBP1-71705) present on the protein construct.
  
  Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20-50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1 ug per load. We recommend starting with 5ug of cell lysate. Add an equal amount of cell lysate and 2X SDS Sample buffer and boil the SDS samples for 10 minutes before loading.
• Theoretical MW: 60 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Store at -80C. Avoid freeze-thaw cycles.
• Buffer: RIPA buffer

Lysate Details for Array

• Type: Overexpression

Notes

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Alternate Names for Tyrosine Hydroxylase Overexpression Lysate

• DYT14
• DYT5b
• EC 1.14.16
• EC 1.14.16.2
• TH
• TYH dystonia 14
• TYH
• Tyrosine 3-hydroxylase
• tyrosine 3-monooxygenase
• Tyrosine Hydroxylase

Background

The tetrameric enzyme tyrosine hydroxylase (TH), also designated as tyrosine 3-monooxygenase (TY3H), is the rate-limiting enzyme for catecholamine biosynthesis and has a theoretical molecular weight of 60 kDa. Encoded by the TH gene, TH catalyzes the conversion of the amino acid L-tyrosine to L-3,4- dihydroxyphenylalanine (L-dopa) in the central nervous system and adrenal medulla. The precursor of catecholamines, L-dopa, is converted to dopamine for use in the biosynthesis of neurotransmitters norepinephrine (noradrenaline) and epinephrine (adrenaline) (1) . Dysfunction in the TH gene, and thus the enzymatic activity by TH, dysregulates catecholamine synthesis.

Two transcription factor binding sites in the proximal region of the TH gene, the TPA-responsive element (TRE) and the c-AMP responsive element (CRE), have been implicated in the complex regulation of the TH gene. Dysregulation of breakdown for the amino acid, tyrosine, by TH is a result of a genetic disorder that results in Tyrosinemia (high levels of tyrosine in the blood, tissue and organs).

Tyrosine hydroxylase deficiency is a disorder that primarily affects movement, where individuals display symptoms that include lack of coordination when walking, postural tremors and unusual body positioning. TH deficient dopamine-responsive dystonia (DRD), also known as Segawa syndrome, is a rare genetic disorder that is associated with low levels of TH and is diagnosed during childhood with characteristic symptoms including increased muscle tone (dystonia) and signs of Parkinsonism like bradykinesia, tremors, rigidity and postural instability (2). Correspondingly, TH is also linked to Parkinson's disease in older adults, where low dopamine levels are a consistent neurochemical abnormality. Functional polymorphisms of the TH gene may be involved in the pathogenesis of neuropsychiatric diseases such as schizophrenia and other affective disorders where dopamine is often dysregulated (3).

References

1. Hamanaka, Y., & Mizunami, M. (2019). Tyrosine hydroxylase-immunoreactive neurons in the mushroom body of the field cricket, Gryllus bimaculatus. Cell Tissue Res, 376(1), 97-111. doi:10.1007/s00441-018-2969-9

2. Li, L., & Zhou, F. M. (2013). Parallel dopamine D1 receptor activity dependence of l-Dopa-induced normal movement and dyskinesia in mice. Neuroscience, 236, 66-76. doi:10.1016/j.neuroscience.2012.12.065

3. Borkar, C. D., Bharne, A. P., Nagalakshmi, B., Sakharkar, A. J., Subhedar, N. K., & Kokare, D. M. (2018). Cocaine- and Amphetamine-Regulated Transcript Peptide (CART) Alleviates MK-801-Induced Schizophrenic Dementia-Like Symptoms. Neuroscience, 375, 94-107. doi:10.1016/j.neuroscience.2018.01.056

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Bioinformatics

• Gene Symbol: TH